Figure 5.

AMSH1 Interacts with ESCRT-III Subunit VPS2.1 through the MIT Domain.

(A) Y2H analysis of GBD-AMSH1 with GAD-fused ESCRT-III subunits VPS2.1, VPS20.1, VPS24.1, and SNF7.1. Transformants were plated on medium lacking Leu, Trp, and His (-LWH) supplemented with 5 mM 3-Amino-1,2,4-triazole (3-AT) (left panel) to test for their auxotrophic growth or on medium lacking Leu and Trp (-LW) (right panel).

(B) Y2H analysis of GBD-AMSH1 (FL; full-length) and GBD-AMSH1(ΔMIT) with GAD-VPS2.1. Transformants were grown on –LWH+6 mM 3-AT (left panel) or -LW (right panel) medium to test for their auxotrophic growth.

(C) In vitro binding assay of the MIT domain of AMSH1 with GST or GST-fused MIM domain of VPS2.1. After GST pulldown, bead-bound proteins were analyzed by immunoblotting using anti-AMSH1 and anti-GST antibodies. Arrowheads indicate the positions of AMSH1(MIT), and asterisks indicate degradation products.

[See online article for color version of this figure.]